Summary

The nuclear factor kappa B (NFκB) pathway controls a variety of processes, including inflammation, and thus, the regulation of NFκB has been a continued focus of study. Here, we report a newly identified regulation of this pathway, involving direct binding of the transcription factor NFκB1 (the p105 subunit of NFκB) to the C-terminus of the A2B adenosine receptor (A2BAR), independent of ligand activation. Intriguingly, binding of A2BAR to specific sites on p105 prevents polyubiquitylation and degradation of p105 protein. Ectopic expression of the A2BAR increases p105 levels and inhibits NFκB activation, whereas p105 protein levels are reduced in cells from A2BAR-knockout mice. In accordance with the known regulation of expression of anti- and pro-inflammatory cytokines by p105, A2BAR-null mice generate less interleukin (IL)-10, and more IL-12 and tumor necrosis factor (TNF-α). Taken together, our results show that the A2BAR inhibits NFκB activation by physically interacting with p105, thereby blocking its polyubiquitylation and degradation. Our findings unveil a surprising function for the A2BAR, and provide a novel mechanistic insight into the control of the NFκB pathway and inflammation.

Introduction

Extracellular adenosine elicits a wide array of physiological and pathological responses via binding to its four subtypes of cell surface receptors A1, A2A, A2B and A3, each of which has a unique pharmacological profile, tissue distribution and effector coupling. Adenosine receptors are typical G protein coupled receptors (GPCRs), which transmit signals through adenylyl cyclase/cAMP and phospholipase C/Ca2+ pathways. It has been well documented that these receptors play important parts in the regulation of inflammation. Depending on the receptor subtype, they may exert anti- or/and pro-inflammatory effects. The A2B adenosine receptor (A2BAR) is a low-affinity adenosine receptor, expressed in immune cells, endothelial cells, aortic vascular smooth muscle, cecum, large intestine and urinary bladder (Yaar et al., 2005). Considering its low affinity in some cases (Schulte and Fredholm, 2000; Schulte and Fredholm, 2003), the A2BAR was hypothesized to play important roles under inflammation and cell stress/damage conditions, where extracellular adenosine is drastically increased (Fredholm, 2007). Interestingly, A2BAR protein level could be selectively upregulated as a result of inflammation, injury, hypoxia and other types of cell stresses (Fredholm et al., 2001; Hart et al., 2009; Kolachala et al., 2005; Kong et al., 2006; Xaus et al., 1999). However, there seems to be no consensus regarding the exact role of A2BAR in the regulation of inflammation (Ryzhov et al., 2008a).

NFκB is a ubiquitously expressed transcription factor regulating various biological functions, including inflammation. Mammalian cells express five NFκB members, including NFκB1 (p50 and its precursor p105), NFκB2 (p52 and its precursor p100), RelA (p65), RelB and c-Rel. Five NFκB inhibitory proteins have been described: IκBα, β and γ as well as p100 and p105 (Hayden and Ghosh, 2004). In resting cells, p105, like other IκB proteins, sequesters NFκB dimers into the cytoplasm. In response to stimulation, p105 is completely degraded by the proteasome, releasing NFκB dimers to translocate into the nucleus and regulate expression of target genes (Pereira and Oakley, 2008). Previous work indicated that p105 plays a pivotal role in immune responses. Deletion of p105 in mice caused severe inflammation and increased susceptibility to opportunistic infections, indicating p105 as a suppressor of inflammation (Ishikawa et al., 1998).

In the present study, we report that the A2BAR directly binds to NFκB1/p105. Our in vitro and in vivo experiments demonstrated that A2BAR binding stabilizes p105 protein by blocking its polyubiquitylation, thereby inhibiting the NFκB signaling pathway. Our study is the first to report that a GPCR directly interacts with p105, thereby identifying a new, unexpected regulator of the NFκB pathway.

Results

In order to better understand A2BAR function, we searched for novel protein binding partners of A2BAR by the yeast two-hybrid (YTH) screening assay, using a human lung cDNA library with the C-terminal 40 amino acids of the A2BAR (A2BAR-C) as a bait. Out of three million screened transformants, 26 positive clones were obtained, of which 20 positive clones encode p105. These results were verified by retransforming yeast with p105 and A2BAR-C (Fig. 1A). Non-identical, overlapping sequences of the positive clones suggested that the high frequency hit was not an artifact of over-amplification of a particular p105 segment in the library, but rather reflected a possible strong interaction of A2BAR and p105.

Identification of p105 as an A2BAR-interacting protein in the YTH system. (A) Yeast colonies co-transformed with pDBLeu-A2BAR-C and pPC86-p105 vectors were first grown on synthetic dropout plates lacking leucine and tryptophan (SD/Leu− Trp−) to ensure successful co-transformation of the two vectors pDBLeu and pPC86, and subsequently were tested for the interaction of p105-A2BAR-C by growing on synthetic dropout plates lacking leucine, tryptophan and histidine, supplemented with 50 µM 3-aminotriazole (SD/Leu− Trp− His− +50 µM 3-AT). pDBLeu-A2BAR-C + pPC86, and pDBLeu + pPC86-p105 are negative controls. (B,C) Mapping the A2BAR-binding region of p105 (B) and the p105-binding region of A2BAR (C) with YTH assays. In B, yeasts were co-transformed with pDBLeu-A2B-C (denoted as A2B-C) and different deletion mutants of p105, as indicated. In C, pPC86-p105 and deletion mutants of A2BAR-C were co-transformed. +, growth; −, no growth. Data shown in A-C are representative of three independent experiments. RHD, Rel homology domain; GRR, glycine-rich region; ANK, ankyrin repeats; DD, death domain; PEST, proline-, glutamic-acid-, serine- and threonine-rich sequence.

To further determine the A2BAR binding site of p105, we constructed various p105 deletion mutants (Fig. 1B). The deletion of the N-terminal amino acids 1–542 or C-terminal PEST domain of p105 completely disrupted its interaction with A2BAR-C in the YTH assay. In contrast, p105 fragments containing amino acids 497–542 and PEST domain bound to A2BAR-C as well as the full-length p105 (Fig. 1B). Thus, the PEST domain and residues 497–542 appear to be essential for the interaction with A2BAR.

In addition, a series of A2BAR-C deletion mutants were constructed to assess the p105 binding site. A2BAR-CΔ292–302 and A2BAR-CΔ292–312 lost their ability to interact with p105 (Fig. 1C). In contrast, the C-terminal deletion mutants A2BAR-CΔ312–332 and A2BAR-CΔ322–332 were still associated with p105 to an extent comparable to the intact A2BAR-C. These data indicated that amino acids 292–302 of A2BAR are necessary, and amino acids 292–312 are sufficient for p105 binding.

Association of A2B adenosine receptor and p105 in mammalian cells

To further confirm A2BAR-p105 interaction, we tested their physical binding by pull-down assays. The full length A2BAR and A2BAR-C were fused to glutathione S-transferase (GST) to generate GST-A2B and GST-A2B-C, respectively. GST-A2B and GST-A2B-C, but not GST alone, efficiently retained endogenous p105 (Fig. 2A, left panel). Reciprocal pull-down assays showed that GST-p105 and GST-p105-C (p105 without p50 region, amino acid residues 433–968), but not GST-p50 or GST alone, pulled down the V5-tagged A2BAR (Fig. 2A, right panel). The results of GST-p105-C are consistent with the yeast mapping data in Fig. 1B.

The association of A2BAR with p105 in mammalian cells. (A) GST pull-down assays. Endogenous p105 in HEK293T cells was pulled down by GST-A2B or GST-A2B-C fusion protein, but not by GST alone (left panel). Data are representative of three independent experiments. In a reciprocal experiment, V5-tagged A2BAR in HEK293T cells was pulled down (denoted as A2B) by GST-p105 or GST-p105-C, but not by GST-p50 or GST alone (right panel). The amounts of GST fusion proteins in each lane are shown at the bottom of each panel. Note that there is also GST-p50 when generating GST-p105, as expected (Beinke and Ley, 2004). Data are representative of four independent experiments. (B) Co-immunoprecipitation assays. Cell lysates from HEK293T cells expressing (+) or not expressing (−) pcDNA4V5-A2BAR (A2B-V5) were immunoprecipitated with anti-V5 antibody. The immunoprecipitates were immunoblotted with anti-p105 or anti-V5 antibodies (left panel). Data are representative of five independent experiments. In a reciprocal experiment, cell lysates from HEK293T cells transfected with Flag-tagged A2BAR and V5-tagged p105 were immunoprecipitated with either mouse anti-V5 or IgG antibodies, followed by immunoblotting with anti-Flag or anti-V5 antibodies (right panel). Data are representative of three independent experiments. Of note, in this right panel, the two different immunoprecipitation experiments involved exactly the same cell lysate (divided into two equal portions) but with two different antibodies (mouse IgG and anti-V5 antibody, and mouse IgG immunoprecipitation was used as an irrelevant immunoprecipitation control). (C) The expression vectors, p105-GFP with CFP alone (upper panel) or A2BAR-CFP (middle row) were transfected into HEK293T cells. After 36 h, the cells were examined by confocal microscopy. In the lower panel, HEK293T cells expressing A2BAR-CFP were fixed and processed for double-staining of p105 and A2BAR with anti-p105 and anti-GFP antibodies, respectively. Scale bar: 5 µm. Data shown are the representative of six independent experiments. ‘Zoom’ views show greater detail of the degree of colocalization of p105 and A2BAR. (D) Co-precipitation of A2BAR and p105 (similar to as shown in B) was not affected by NECA. At 24 h after transfection of V5-tagged A2BAR, cells were treated with A2BAR agonist 10 µM NECA or DMSO (vehicle) for 30 min. Cell lysates were then immunoprecipitated with anti-V5 antibody. Data are the representative of three independent experiments. Note that the p105 level increases in the presence A2BAR in panels B and D.

A2BAR-p105 interaction was further verified by co-immunoprecipitation assays in HEK293T cells, using a tagged-A2BAR because of the lack of a reliable anti-A2BAR antibody (supplementary material Fig. S1). Cell extracts of HEK293T cells transfected with V5-tagged A2BAR were immunoprecipitated with an anti-V5 antibody. Endogenous p105 coprecipitated with A2BAR, whereas no immunoreactive material was detected from cells transfected with mock vector pcDNA4-V5 (Fig. 2B, left panel). In a reciprocal experiment, V5-tagged p105 was expressed with Flag-tagged A2BAR in HEK293T cells, followed by immunoprecipitation. A2BAR was co-immunoprecipitated by anti-V5 antibody but not by control mouse IgG (Fig. 2B, right panel). Taken together, these results indicated that the A2BAR and p105 are also capable of forming a complex in mammalian cells. In addition, the co-localization of A2BAR and p105 was analyzed by confocal microscopy, in living cells to avoid shrinkage of the cytoplasm during the cell fixation process and therefore better assess the intracellular localization of A2BAR, and in fixed cells to examine endogenous p105. A2BAR-CFP was localized in both the plasmalemma and cytoplasm (Fig. 2C), as has been observed for some other GPCRs (Wang et al., 2005). Importantly, A2BAR-CFP were co-localized with p105-GFP or endogenous p105 in both the plasmlemma and cytoplasm (Fig. 2C), consistent with the idea that A2BAR and p105 physically interact in the cell.

Next, we asked whether A2BAR-p105 association is dependent on A2BAR agonist stimulation. At 24 h after transfection of V5-tagged A2BAR, cells were treated with 10 µM N-ethyl carboxamidoadenosine (NECA), an A2-type adenosine receptor agonist. NECA, although eliciting a robust elevation of cAMP level (supplementary material Fig. S2), had little effect on A2BAR-p105 interaction, arguing for an agonist-independent A2BAR and p105 coupling (Fig. 2D). It was noted that p105 protein level markedly increased in the presence of A2BAR (Fig. 2B,D, also see Fig. 4).

A2BAR stabilized p105 and p50 protein expressions in vitro. (A) HEK293T cells were transfected with V5-tagged A2BAR (400 ng plasmid), and after 36 h, p105, p50, p65, A2BAR and β-actin protein levels were measured in whole cell lysates by immunoblotting (upper panel). p105-encoding transcripts were analyzed by RT-PCR (middle panel). The lower panel shows the quantification of the relative levels of mRNA encoding p105 in four experiments that were similar to that shown in the middle panel. (B) Immunoblotting, with the indicated antibodies, of the lysates of HEK293T cells transfected with increasing doses of V5-tagged A2BAR (with a maximum of 400 ng plasmid). (C) At 24 h after transfection of V5-tagged A2BAR (100 ng plasmid), HEK293T cells were treated with 10 µM NECA or DMSO (vehicle) for 12 h, then cell lysates were collected for immunoblotting of p105, p50 and p65 protein expression. Note the relatively small changes of p105 and p50 because there was less A2BAR expression (100 ng) in these experiments than those shown in panel A and B (400 ng A2BAR). Data shown in panels A-C are the representative of 4–5 independent experiments. (D) At 6 h after transfection of A2BARSiRNA or NcSiRNA (control), HA-tagged IKKβ-SS/EE or mock vectors (pcDNA3.1-HA) were expressed in HEK293T cells. The expression of various proteins was examined with immunoblotting, and the expression of endogenous A2BAR was examined by RT-PCR rather than immunoblotting because of lack of reliable A2BAR antibodies (upper panel). The lower panel shows a summary of the relative protein amount of p105 normalized to β-actin in six experiments that were similar to those shown in the upper panel. *significantly different from NcSiRNA, P = 0.010 for cell without IKKβ-SS/EE, **P = 0.008 for cell with IKKβ-SS/EE. β-actin was used as a loading control in panels A-D. (E) V5-tagged wild-type A2BAR, mutant A2BAR (A2BARΔ292–302) or pcDNA4-V5 (Mock) expressed in HEK293T cells was immunoprecipitated with anti-V5 antibody. The immunoprecipitates were immunoblotted with anti-p105 or anti-V5 antibodies. Whole cell lysates were analyzed with anti-p105 and β-actin antibodies (upper panel). The lower panel shows quantification of the relative protein amount of p105 normalized to β-actin in five experiments similar to those shown in the upper panel. **different from Mock cells, P = 0.0014.

Direct interaction of A2BAR with p105

Although GST pull-down, co-immunoprecipitation and co-localization assays detected an association of A2BAR and p105, these assays are incapable of distinguishing between direct protein-protein interactions and those mediated by an additional protein(s). Early studies suggested that β-arrestin physically interacts with p105 (Parameswaran et al., 2006) or A2BAR (Matharu et al., 2001). To address whether p105-A2BAR association is mediated by β-arrestin or not, β-arrestin-null murine embryonic fibroblasts (MEFs) were used (supplementary material Fig. S3). Importantly, p105 interacted with the A2BAR even in β-arrestin-null MEFs (Fig. 3A, right panel). This observation demonstrated that β-arrestin is not necessary for p105-A2BAR binding, although it does not rule out the involvement of β-arrestin in p105-A2BAR interaction. To further exclude the presence of additional protein mediators of p105 and A2BAR interaction, GST-p105 or GST-p105-C and maltose-binding protein (MBP)-tagged A2BAR-C or MBP alone were purified from E. coli (supplementary material Fig. S4) and then used for pair-wise GST pull-down assays. Results showed that MBP-tagged A2BAR-C, but not MBP alone, binds to p105 or p105-C (Fig. 3B; supplementary material Fig. S5), further strengthening the conclusion that A2BAR-C-p105 interaction is independent of accessory protein(s).

Direct interaction of p105 with the A2BAR. (A) Association of A2BAR with p105, both in β-arrestin1/2 wild-type (WT) and knockout (KO) MEF cells. β-arrestin1/2 WT or KO MEF cells were transfected with V5-tagged A2BAR. After 36 h, cell lysates were incubated with purified GST, GST-p50, GST-p105-C or GST-p105 fusion protein (30 µg each). A2BAR was pulled down by GST-p105 or GST-p105-C, but not by GST-p50 or GST alone in both β-arrestin1/2 wild-type (left panel) and knockout MEF cells (right panel). Data shown are the representative of four independent experiments. (B) GST, GST-p50, GST-p105 or GST-p105-C fusion protein was immobilized on glutathione beads and incubated with purified MBP-tagged A2B-C protein. Pulled-down proteins were then visualized by immunoblotting with anti-MBP antibodies. The loading of purified MBP-A2B-C and GST fusion proteins is also shown. Data shown are the representative of three independent experiments.

Remarkably, the two A2BAR binding sites of p105 (Fig. 1B) overlap with the potential ubiquitin ligase binding sites (Beinke and Ley, 2004; Orian et al., 1999). We reasoned that A2BAR binding may interfere with p105 ubiquitylation and therefore regulate its stabilization. Indeed, ectopic expression of the A2BAR dramatically increased endogenous p105 protein, while it had little impact on p105 mRNA level (Fig. 4A, also Fig. 2B,D), indicating a post-transcriptional regulation. A2BAR expression increased p105 protein expression in a dose-dependent manner (Fig. 4B). It was also noted that A2BAR expression was accompanied by a reproducible increase in the level of p50 (Fig. 4A–C), presumably secondary to augmented levels of p105 protein, which is processed into p50 (Beinke and Ley, 2004). It has been well known that the overexpression of GPCRs without agonist stimulation mimics agonist-dependent activation of the receptors (Milano et al., 1994). To exclude the possibility that the stabilizing effect of A2BAR on p105 protein expression results from A2BAR activation by A2BAR overexpression, or by minimal adenosine present in the culture media, we examined the effect of NECA on p105 expression. NECA (10 µM) had no effect on p105 protein level (Fig. 4C; supplementary material Fig. S6), indicating that A2BAR expression stabilizes p105 in an agonist-independent manner. In another set of experiments, we assessed p105 protein level after knocking down endogenous A2BAR by siRNA. Knockdown of endogenous A2BAR robustly reduced endogenous p105 protein level in resting cells (Fig. 4D), but had no effect on p105 mRNA level (supplementary material Fig. S7).

In the canonical NFκB pathway, IκB kinase β (IKKβ) induces quick degradation of p105 and activates NFκB signaling (Beinke and Ley, 2004). We, thus, assessed A2BAR-mediated p105 stabilization after p105 degradation was accelerated by the expression of IKKβ-SS/EE, the constitutively active form of IKKβ. The level of p105 was reduced by IKKβ-SS/EE expression, as expected, and further decreased by A2BAR knockdown (Fig. 4D). Because amino acids 292–302 of A2BAR are necessary for binding to p105 in YTH assays (Fig. 1C), we generated a binding deficient mutant (A2BARΔ292–302) of A2BAR by deleting amino acids 292–302. As expected, the mutation abolished the interaction of A2BAR and p105 in HEK293T cells (Fig. 4E). More importantly, the mutant failed to increase, even slightly decreased, p105 protein expression (Fig. 4E), demonstrating that the physical association with p105 is critical for the stabilizing effect of A2BAR on p105.

In view of the fact that A2BAR and ubiquitin ligases binding sites in p105 overlap (Fig. 1B), we tested whether A2BAR binding obstructs p105 ubiquitylation and thereby prevents ubiquitin-proteasome dependent degradation of p105. Significantly, A2BAR expression markedly suppressed p105 polyubiquitylation (Fig. 5A). MG132, the proteasome inhibitor, greatly elevated p105 polyubiquitylation, confirming that polyubiquitylated p105 is mostly, if not all, directed to the proteasome for degradation. In marked contrast, MG132 had little effect on p105 polyubiquitylation in the presence of A2BAR expression, further supporting that the A2BAR effectively blocks the proteasome-targeted ubiquitylation of p105. As control, the expression of another integral membrane protein cystic fibrosis transmembrane conductance regulator (CFTR) slightly increased p105 ubiquitylation, arguing against the possibility that the decrease in p105 polyubiquitylation upon A2BAR expression is an artifact resulting from protein overexpression. It is well known that IKKβ phosphorylates serines 927 and 933 in p105, triggering the ubiquitylation and subsequent degradation of p105. To test whether A2BAR binding also affects p105 phosphorylation, we compared the phosphor-p105 (Serine 933) level in the presence or the absence of A2BAR. A2BAR had no effect on the phosphorylation level of p105 (Fig. 5B), suggesting that A2BAR specifically blocks the binding of ubiquitin ligases, but not IKKβ, to p105.

A2BAR expression suppressed p105 polyubiquitylation but not phosphorylation. (A) HEK293T cells expressing Flag-tagged ubiquitin and V5-tagged p105 with or without CFP-tagged A2BAR or GFP-tagged CFTR. At 48 h after transfection, cells were treated with 10 µM MG132 or DMSO (vehicle) for 3 h, followed by immunoprecipitation of p105 with anti-V5 antibodies. Immunoblotting of polyubiquitylated p105 (upper panel) and corresponding immunoprecipitated p105 (second panel) are shown. Note that loading of p105 in the two lanes with A2B-CFP expression are only 40% of other lanes in order to better view the differences in p105 ubiquitylation. Summary data of the relative ubiquitylation amount of p105 (normalized to the amount of immunoprecipitated p105 protein) were shown in the lower panel. *significantly different from Mock cells (pEGFP-N1 transfected cells), P = 0.021 for DMSO, P = 0.026 for MG132 (n = 4). (B) HEK293T cells were transfected with V5-tagged A2BAR or GFP-tagged CFTR or pcDNA4-V5 (Mock) in the presence or absence (control) of HA-tagged IKKβ-SS/EE. After 36 h, the levels of total and phosphorylated p105, and other various proteins were subjected to immunoblot analysis (upper panel). The summary data of the relative phosphorylation of p105 (normalized to the total amount of p105 protein) is shown in the lower panel (n = 4; P>0.05).

p105 acts as a NFκB-inhibitory protein, retaining p50, p65 or c-Rel in the cytoplasm and inhibiting NFκB signaling pathway. Our data showed that A2BAR/p105 complex could interact with p65 and p50 (supplementary material Fig. S8). To examine the effect of A2BAR-p105 interaction on NFκB activation, HEK293T cells were first transiently co-transfected with the V5-tagged A2BAR and an NFκB-dependent luciferase reporter gene. A2BAR expression modestly suppressed basal NFκB transcription activity, but this effect was much more profound in the presence of IKKβ-SS/EE stimulation (Fig. 6A, left panel, see Discussion). CFTR, as a control, failed to alter NFκB activation. Moreover, NFκB activation was significantly increased by knocking down endogenous A2BAR with siRNA treatment under both basal and IKKβ-SS/EE induced conditions (Fig. 6A, right panel), supporting that endogenous A2BAR expression inhibits NFκB signaling. These observations are further strengthened by the nuclear translocation assays using TransAM NFκB ELISA kit. Ectopic expression of the A2BAR inhibited p65 and p50 nuclear translocation, especially with IKKβ-SS/EE stimulation, while knocking down endogenous A2BAR increased the nuclear translocation of p65 and p50 with or without IKKβ-SS/EE. As control, A2BAR did not change the nuclear translocation level of AP1 transcription factor c-Fos (Fig. 6B). It is noted that in the absence of IKKβ-SS/EE stimulation the inhibitory effect of ectopic A2BAR expression on p65 and p50 nuclear translocation is small and not statistically significant, presumably because of the limited sensitivity of the assay.

p105 expression is reduced in A2BAR−/− mice

Next, we examined the interrelation between A2BAR and p105 expression in mice. We previously reported that the A2BAR is expressed primarily or only in the vasculature of tissues, such as the pancreas, lung, and spleen. This was traced using β-galactosidase (β-gal) staining, as our A2BAR knockout mice include β-gal-encoding gene under the control of the A2BAR gene promoter, instead of the deleted A2BAR gene (Yang et al., 2006). High A2BAR expression was noted in arteries (Yang et al., 2006). In accordance, A2BAR deficiency is associated with reduced levels of p105 in the mesenteric artery (Fig. 7A), but it does not induce a significant change in the basal level of p50 or p65 (supplementary material Fig. S9A). Macrophages are also a significant site of expression of this receptor (Yang et al., 2006). In these cells too p105 level is reduced compared to wild type (Fig. 7B), with no significant changes in p50 or p65 (supplementary material Fig. S9B). Finally, A2BAR deficiency does not promote a change in the level of p105 in various tissues, which typically do not express significant levels of the A2BAR (supplementary material Fig. S9C). Lipopolysaccharide (LPS) upregulates the expression of the A2BAR in various tissues (St Hilaire et al., 2008; and data not shown). Here, we show that p105 is downregulated in different tissues following in vivo administration of LPS into A2BAR knockout mice, relative to wild-type mice (Fig. 7C). As control, p65, p50 and IκBα expression levels did not change significantly between the control and KO samples. Taken together, these results are in line with our above-described biochemical studies, which showed that A2BAR expression stabilizes the level of p105 protein.

Reduced levels of p105 in macrophages and tissues from A2BAR knockout mice. (A,B) Under basal conditions, total proteins isolated from the mesenteric artery (a site of high A2BAR expression; panel A) and peritoneal macrophages (non activated; see Materials and Methods; panel B) of wild-type (WT) and A2BAR knockout (KO) mice were used. Representative immunoblotting results with anti-p105 antibody are shown in the upper panels, with β-actin used as loading control. The lower panels of show the quantification of western blots. In the mesenteric artery, p105 values for KO are significantly different from WT, ***P = 0.0006 (n = 6). Similarly, in macrophages the differences between WT and KO are statistically significant; **P = 0.0095 (n = 3, each run twice). (C) Wild-type and A2BAR KO mice were injected intraperitoneally with LPS (10 mg/kg body weight). Proteins from various tissues were collected after 5 h and immunoblotted (upper panel). The lower panel shows the quantification of p105 expression in eight experiments similar to that shown in the upper panel. ***significantly different from WT A2BAR mice, P = 0.001 for thymus; *P = 0.038 for lung; **P = 0.007 for spleen; *P = 0.020 for pancreas.

Cytokines production in A2BAR−/− mice

The expression of target genes of NFκB signaling was examined as additional functional evidence of reduced p105 expression in A2BAR knockout mice. Numerous genes are modulated by NFκB signaling, however, we primarily focused on cytokine gene expression in order to better understand the role of A2BAR in inflammation. Previous studies indicated that NFκB signaling regulates the production of numerous cytokines, including TNF-α, IL-2, IL-6, IL-8 and IL-12 (Blackwell and Christman, 1997). In addition, it was suggested that following LPS treatment p105/p50 deficient mice produce more pro-inflammatory cytokines, such as TNF-α and IL-12, and less anti-inflammatory cytokines, including IL-10 (Cao et al., 2006). Therefore, to define the impact of A2BAR expression on p105-mediated NFκB signaling, we measured serum concentrations of TNF-α, IL-12 and IL-10 in LPS-treated A2BAR knockout mice. A2BAR deficiency resulted in 31% suppression of IL-10 and 35% elevation of TNF-α at 1 h, and 35% augmentation of IL-12 at 5 h after LPS stimulation (Fig. 8). These results are consistent with data reported by Yang et al. (Yang et al., 2006) and support the notion that A2BAR expression, in association with elevated p105 (Fig. 7), regulates cytokine production, thereby suppressing inflammatory responses. Since adenosine is present at some level in different cellular milieus, we cannot conclude that some or all these effects in vivo are agonist-independent.

Discussion

Our study has unveiled a novel mechanistic insight into the control of the NFκB pathway and inflammation, and a surprising function of A2BAR, a typical member of the GPCR family. This study is the first to report that a GPCR directly interacts with p105. We have shown that A2BAR binds to p105 in regions overlapping with the ubiquitin ligase binding sites, and therefore sequesters p105 from polyubiquitylation and proteosome-dependent degradation, resulting in downregulation of NFκB signaling in an agonist-independent way. This agonist-independent A2BAR regulation of NFκB signaling may have important biological implications, given that inflammation and cell stress selectively upregulate A2BAR expression by 3–4 folds (Blackburn et al., 2009). The elevation of A2BAR expression induced by inflammation could downregulate NFκB signaling and suppress inflammation, forming a negative feedback loop (summarized in supplementary material Fig. S10).

Emerging evidence suggests that GPCRs can also signal through G-protein independent pathways, although the classical paradigm of GPCR signaling involves interaction of ligand with the receptor, followed by G protein activation by the ligand-bound receptor and modulation of intracellular signaling proteins, or targets. The effectors of the G-protein independent pathways include ion channels, and c-Src and Jak kinases (Liu et al., 2000).

Although the present study is the first report of a Family A GPCR directly interacting with a transcriptional factor, two independent studies have previously found that the metabotropic GABAB receptor, a Family C GPCR, directly interacts with the transcription factors ATF4 (CREB2) and ATFx to modulate their cytosol-to-nucleus translocation, even though in an agonist- and G-protein-dependent manner (Nehring et al., 2000; White et al., 2000). In addition, GABAB receptor also interacts with the transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), resulting in reduced surface expression of the receptor (Sauter et al., 2005). In our study, we addressed whether the physical and functional interaction between A2BAR and p105 depends on A2BAR agonist, as the interactions of GABAB and β2 adrenergic receptors with transcriptional factors are agonist-dependent (Gao et al., 2004; White et al., 2000). Our data support an agonist-independent mechanism. Of note, however, in our experiments, we examined the effect of A2BAR activation by NECA on the interaction of A2BAR and p105, but not on NFκB activation per se. Our data suggest that NECA did not affect NFκB by altering the interaction of A2BAR and p105, but the current study did not test whether or not the activation of A2BAR could stimulate NFκB through other signaling pathways. We also examined the effect of A2BAR-p105 interaction on A2BAR function, considering that its interaction with the transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) resulted in reduced surface expression of GABAB receptor (Sauter et al., 2005). p105 expression did not seem to have any impact on A2BAR-mediated cAMP generation (not shown).

It is well documented that GPCRs regulate NFκB signaling via activation of PKA, PKC and small GTPases (Diaz-Meco et al., 1994; Zhong et al., 1998). A GPCR has recently been linked with IκBα and NFκB/p105 through arrestin (Gao et al., 2004; Parameswaran et al., 2006; Witherow et al., 2004), which binds to ligand-bound GPCRs and mediates receptor endocytosis. Among the four isoforms of the arrestin family, two are expressed exclusively in the visual system, and the other two (β-arrestin 1 and 2) are ubiquitously expressed. It was suggested that arrestin directly bind to the DD/PEST domains of IκBα (Gao et al., 2004; Witherow et al., 2004) or p105 (Parameswaran et al., 2006) and prevents their phosphorylation and degradation. Interestingly, agonist stimulation of β2 adrenergic receptor enhances the interaction of arrestin and IκBα (Gao et al., 2004). However, this effect could be mimicked neither by the stimulation of other GPCRs, such as δ opioid receptor and bradykinin receptor, nor by stimulation of adenylyl cyclase (Gao et al., 2004). The mechanism underlying this receptor-specific effect remains unknown. In view of these observations, we asked whether A2BAR-p105 association is a direct protein-protein interaction or is also bridged by β-arrestin. By using β-arrestin null MEF cell lines, we demonstrated that the A2BAR is able to bind to p105 directly.

The role of the A2BAR in inflammation has been seemingly puzzling. Numerous pharmacological studies demonstrate that the activation of A2BAR by adenosine triggers proinflammatory effects by upregulating proinflammatory cytokines and growth factors and downregulating anti-inflammatory cytokines in many cell types (Donoso et al., 2005; Feoktistov and Biaggioni, 1995; Fiebich et al., 1996; Rees et al., 2003; Ryzhov et al., 2004; Ryzhov et al., 2008a; Zhong et al., 2004), while there is also some contradictory evidence, depending on the systems and conditions used (Eckle et al., 2008a; Eckle et al., 2008b). Yang and colleagues showed that A2BAR knockout mice display mild elevation of proinflammatory TNF-α and IL-6, and decreased levels of anti-inflammatory IL-10 in the plasma under basal conditions, and more so under LPS stimulation. This was associated with upregulation of the NFκB pathway (Yang et al., 2006). These findings implied that the A2BAR is constitutively activated by the basal concentration of adenosine. This was rather puzzling, considering that in some cellular systems the A2BAR is only activated by high adenosine levels, occurring during cell/tissue stress and damage (Fredholm, 2007). The observation of Yang et al. (Yang et al., 2006), pointing to an anti-inflammatory role of A2BAR under basal conditions, was supported by a number of following studies in A2BAR knockout mice (Csóka et al., 2010; Eckle et al., 2008a; Eckle et al., 2008b; Frick et al., 2009; Grenz et al., 2008; Hart et al., 2009; Hua et al., 2007; Ryzhov et al., 2008b; Yang et al., 2008; Zhou et al., 2009). However, most of these studies did not scrutinize whether the basal phenotype in A2BAR knockout mice resulted from loss of direct agonist activation, or rather an unidentified function of the A2BAR. Ryzhov et al., (Ryzhov et al., 2008a) observed an increase in inflammatory indices under basal conditions (non LPS) in A2BAR knockout mice compared to the wild type in the absence of adenosine stimulation, similar to the studies by Yang et al. (Yang et al., 2006) and other groups. Similar to the conclusion of these authors, we deduced that the A2BAR can also exert an adenosine-independent downregulation of pro-inflammatory cytokines by associating with a previously unrecognized signaling pathway(s), identified in our current study. We also consider the report of direct regulation of expression of IL-10 by NFκB (Cao et al., 2006). Our data suggest an alternative explanation of how the A2BAR could regulate IL-10 production through A2BAR-dependent NFκB signaling, in addition to the A2BAR-p38-CREB pathway reported by Koscso et al. (Koscsó et al., 2012).

In our in vivo experiments, no significant change in the basal protein level of p105 was observed in tissues from A2BAR knockout mice in which the A2BAR is typically poorly expressed, compared with wild-type mice. It is also possible that in in vivo resting cells p105 is “inactive” and sequesters NFκB dimer in the cytoplasm, and therefore, the protective effect of A2BAR is not manifested. LPS effectively upregulates the expression of the A2BAR (St Hilaire et al., 2008). Upon LPS stimulation, most p105 is degraded in A2BAR knockout mice, whereas in wild-type mice p105 levels are high, which would be expected if the A2BAR prevents p105 polyubiquitylation and degradation. Of note, luciferase activity and nuclear translocation of p65 or p50, indicative of NFκB signaling was modestly downregulated upon ectopic expression of the A2BAR, although p105 level was augmented. This is probably due to already saturating levels of p105 in the control cells prior to A2BAR overexpression. Alternatively, the overexpression of A2BAR activates the receptors without agonists, as it is well known that the overexpression of GPCRs without agonist stimulation mimics agonist-dependent activation of the receptors (Milano et al., 1994). The activation of A2BAR could stimulate NFκB signaling via PKA and PKC (Diaz-Meco et al., 1994; Zhong et al., 1998), masking its agonist-independent protective effect on p105 in unstimulaled cells, and this masking effect is likely trivial when NFκB signaling is substantially activated by IKKβ.

We consistently observed elevation of p50 level with p105 protein increase as a result of ectopic A2BAR expression (Fig. 4A,B). However, p105 decrease associated with either A2BAR knock down/knockout or IKKβ stimulation was not accompanied with a p50 decrease (Fig. 4D; Fig. 7C; supplementary material Fig. S9). It has been previously reported that decrease of p105 was not associated with any changes in basal p50 level (Heissmeyer et al., 2001), suggesting that basal p50 is primarily generated through de novo synthesis (with co-translation of p105) (Ciechanover et al., 2001) and p105 processing into p50 is largely inhibited with docking of p50 under basal conditions (Cohen et al., 2001). However, when p105 is upregulated post-translationally without accompanying p50 increase from co-translation, pre-existing level of p50 may not be sufficient to block p105 processing into p50 and therefore an increase of p50 will occur.

Finally, initial co-immunoprecipatation experiments indicated that p105 does not bind to the A2AAR (data not shown). However, we cannot rule out the possibility that p105 interacts with other members of the adenosine receptor family. This could constitute a future line of investigations.

In sum, our study unveils novel mechanistic insights into the control of the NFκB pathway and inflammation. It is the first report to show that the A2BAR negatively regulates NFκB activation by physically interacting with p105, thereby blocking its polyubiquitylation and degradation, with clear implications on NFκB-regulated pathways (supplementary material Fig. S10).

Materials and Methods

Mice

A2BAR knockout mice were generated in an earlier study (Yang et al., 2006) and were backcrossed to C57BL/6J mice to reach a pure C57BL/6 background (Yang et al., 2008). Wild-type and A2BAR knockout mice were strain-, sex-, and age-matched (6–8 weeks old, unless otherwise indicated). All animal procedures were approved by the University Committee on Research Practices at Hong Kong University of Science and Technology.

RNA isolation and RT-PCR

Total RNA was isolated from HEK293T cells with TRIZOL® reagents (Invitrogen). RT-PCR was performed with OneStep RT-PCR kits (Qiagen). The primer pairs used were as follows: for A2BARs, 5′-CTCTTCCTCGCCTGCTTCGT-3′ (sense) and 5′-GGGCAGAACACACCCAAAGAA-3′ (antisense) (expecting a 345 bp fragment); for p105, 5′-ATGGCAGAAGATGATCCA-3′ (sense) and 5′-AAATTTTGCCTTCTAGAGG-3′ (antisense) (expecting a 2.9 kb fragment). β-actin was used as a loading control. The identities of the PCR products were confirmed by DNA sequencing.

RNA interference

RNA oligonucleotides were synthesized by Dharmacon (Lafayette, CO). The A2BAR siRNA 5′-AACCGAGACUUCCGCUACA-3′or non-targeting siRNA (Catalog no. D-001210-01-05) was inserted into pSuper vector (Oligoengine, Seattle, WA). HEK293T cells grown on plates at 70–80% confluency were pre-incubated for 6 h with A2BAR or non-targeting siRNA and Lipofectamine 2000 (Invitrogen) in FBS-free DMEM. The cells were then transfected with other various vectors and grown for 36–48 h before collection.

Pull down assays

After bound with GST or GST fusion proteins generated and purified from E. coli strain BL21, the glutathione Sepharose beads were loaded with cell lysates at 4°C overnight, followed by 3 washes. The bound proteins were analyzed by western blotting. MBP-A2B-C fusion protein was expressed in BL21 bacteria and purified by amylose resin kit (New England Biolabs).

Ubiquitylation assays

At 48 h after transfection, HEK293T cells were pretreated with MG132 (10 µM) or DMSO for 3 h at 37°C and lysed for 40 min in ubiquitylation lysis buffer A (8 M urea, 100 mM NaH2PO4, 1% Triton X-100, 10 mM Tris, pH 8.0). The supernatant was incubated with Ni2+-nitrilotriacetic acid-agarose (Qiagen) for 2 h. After washing twice with buffer A and twice with buffer B (same as buffer A except for 0.5% Triton X-100 and pH 6.3), the proteins were eluted twice with buffer C (same as buffer A except for 0.1% Triton X-100 and pH 4.5). All the procedures were performed at room temperature.

NFκB and AP1 activation assays

Nuclear proteins from HEK293T cells were prepared using the TransAMTM transcription assay kit (Active Motif). Nuclear extracts (5 µg; prepared as instructed by the manufacturer) were used to measure p65 and p50 nuclear translocation with TransAMTM NFκB Family kit, following the manufacturer's protocol. Nuclear c-Fos was analyzed with the TransAMTM AP1 c-Fos kit (Active Motif).

Mouse tissue isolation and cytokine assays

Mesenteric arteries were removed from A2BAR KO and control 8-week-old male mice (strain-matched) and flash frozen. The tissue was homogenized and lysed on ice in radioimmunoprecipitation assay (RIPA) buffer A (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with proteinase inhibitor cocktail. Lysates were vortexed for 10 min at 4°C and then placed on ice for 30 min. The lysates were then frozen in liquid nitrogen and then thawed at 37°C. This freeze-thaw cycle was repeated. The samples were centrifuged at 800 g for 10 min at 4°C and the infranatant removed. This was repeated to clear the sample of all cell debris and remaining fat tissue. Following protein determination with the BioRad assay, samples were subjected to western blotting (75 µg/lane). To upregulate the expression of the A2BAR in other tissues, 6–8-week-old A2BAR KO and wild-type mice were challenged with Lipopolysaccharides (LPS) administered intraperitoneally (10 mg/kg, Sigma 026:B6). For immunoblotting, mice were sacrificed 5 h after LPS treatment, different tissues were homogenized in RIPA buffer B (50 mM Tris-HCl, 1.0 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate, 1 mM PMSF, pH 7.4). After centrifugation at 16,000 g for 10 min, the supernatant was collected for immunoblot analysis. For cytokine assays, mouse serum was collected at different time points from 1–10 h after LPS challenge, a time frame in which cytokine release is maximal (Cao et al., 2006). The levels of TNFα, IL-10, and IL-12 were measured using ELISA kits from eBioscience (San Diego, CA).

Peritoneal macrophage isolation

Peritoneal macrophages were collected from 12-week-old mice as non activated and seeded into plates at 1×106 cells/ml in Macrophage serum free medium (Invitrogen, 12065), supplemented with 0.1% penicillin-streptomycin for 2 hours at 37°C. Cells were then collected in RIPA buffer A (see above) for western blot analysis. This yields a low number of macrophages, all used for western blotting (50 µg protein/lane).

Quantification of western blots and statistics

Quantification of protein bands in western blots was carried out with ImageJ software. All data are expressed as means ± SE. Unless indicated otherwise, Student's two-tailed t-test was used for statistical analysis. P<0.05 was considered as statistically significant.

Acknowledgments

We thank Dr Robert J. Lefkowitz (Duke University) and Dr Gang Pei (Shanghai Institutes for Biological Sciences Chinese Academy of Sciences) for kindly providing β-arrestin WT and KO MEF cell lines, Dr Shanthi V. Sitaraman (Emory University) for A2BAR cDNA, Dr Gerd Walz (University of Freiburg) for flag-tagged ubiquitin vector, and Dr Xiandi Gong (Nanyang Technological University) for pIRES2-EGFP-CFTR vector. We are also grateful to Mr Kalun So and Ms Quan You Li for technical assistance and Dr Wei Zhang for help in figure preparation. Y.S. and P.H. conceived the study and wrote the manuscript; P.H. directed the study; Y.S. performed and analyzed most of the work; Y.D. performed initial yeast two-hybrid screening and contributed to experimental design and data analysis in other yeast two-hybrid assays, A.S.E. performed analyses in mouse mesenteric artery and macrophages; W.H. performed macrophage assays; A.Q. performed initial yeast two-hybrid screening; W.K.L. performed pair-wise pull down assays; Y.W. partially contributed to ubiquitylation and A2BAR knock down experiments; W.Z. provided reagents and contributed to experimental design; K.R. provided the A2BAR knockout mice, participated in the analyses of arteries and macrophages, provided conceptual insights, and participated in manuscript writing.

Footnotes

Funding

This work was supported by Hong Kong Research Grants Council [grant number GRF661008 to P.H.]; and by the National Heart, Lung, and Blood Institute (NHLBI) [grant number HL093149 to K.R.]. Deposited in PMC for release after 12 months.

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